Randall Thompson

Dominant-negative suppression of Cav2.1 currents by α12.1 truncations requires the conserved interaction domain for β subunits

Abstract Episodic ataxia type 2 (EA2) is an autosomal dominant disorder arising from CACNA1A mutations, which commonly predict heterozygous expression of Ca v 2.1 calcium channels with truncated α 1 2.1 pore subunits. We hypothesized that α 1 2.1 truncations in EA2 exert dominant-negative effects on the function of wild-type subunits. Wild-type and truncated α 1 2.1 subunits with fluorescent protein tags were transiently co-expressed in cells stably expressing Ca v auxiliary β subunits, which facilitate α 1 subunit functional expression through high-affinity interactions with the alpha interaction domain (AID). Co-expression of wild-type subunits with truncations often resulted in severely reduced whole-cell currents compared to expression of wild-type subunits alone. Cellular image analyses revealed that current suppression was not due to reduced wild-type expression levels. Instead, the current suppression depended on truncations terminating distal to the AID. Moreover, only AID-bearing α 1 2.1 proteins co-immunoprecipitated with Ca v β subunits. These results indicate that Ca v β subunits may play a prominent role in EA2 disease pathogenesis.

Differential induction of indoleamine-2,3-dioxygenase (IDO) by interferon-γ in human gyneocologic cancer cells

Abstract Induction of indoleamine-2,3-dioxygenase (IDO) by interferon-γ (IFN-γ) is thought to be one mechanism underlying IFN-γs antineoplastic properties. Since clinical trials with IFN-γ have yielded variable efficacy in treating cancers of gynecological origin, we tested the effects of IFN-γ on cell growth and IDO activity in cell lines from seven gynecologic and five breast cancers. At a dose of 250 IU/ml, IFN-γ suppressed cell growth and induced IDO activity in one cervical (C41), one vulva (A431), one breast (HS578T) and two ovarian (OVCAR-3, CAOV-3) cancer cell lines. Differing inhibition of cell growth, but with no induction of IDO activity, was found with IFN-γ treatment of the other cell lines.

Trigeminal interpolaris/caudalis transition neurons mediate reflex lacrimation evoked by bright light in the rat

Abnormal sensitivity to bright light can cause discomfort or pain and evoke protective reflexes such as lacrimation. Although the trigeminal nerve is probably involved, the mechanism linking luminance to somatic sensory nerve activity remains uncertain. This study determined the effect of bright light on second‐order ocular neurons at the ventral trigeminal interpolaris/caudalis transition (Vi/Vc) region, a major termination zone for trigeminal sensory fibers that innervate the eye. Most Vi/Vc neurons (80.9%) identified by responses to mechanical stimulation of the ocular surface also encoded bright light intensity. Light‐evoked neural activity displayed a long latency to activation (> 10 s) and required transmission through the trigeminal root ganglion. Light‐evoked neural activity was inhibited by intravitreal injection of phenylephrine or l‐NG‐nitro‐arginine methyl ester (L‐NAME), suggesting a mechanism coupled to vascular events within the eye. Laser Doppler flowmetry revealed rapid light‐evoked increases in ocular blood flow that occurred prior to the increase in Vi/Vc neural activity. Synaptic blockade of the Vi/Vc region by cobalt chloride prevented light‐evoked increases in tear volume, whereas blockade at the more caudal spinomedullary junction (Vc/C1) had no effect. In summary, Vi/Vc neurons encoded bright light intensity and were inhibited by drugs that alter blood flow to the eye. These results support the hypothesis that light‐responsive neurons at the Vi/Vc transition region are critical for ocular‐specific functions such as reflex lacrimation, whereas neurons at the caudal Vc/C1 junction region probably serve other aspects of ocular nociception.

Sensitization of trigeminal brainstem pathways in a model for tear deficient dry eye

Abstract Chronic dry eye disease (DE) is associated with an unstable tear film and symptoms of ocular discomfort. The characteristics of symptoms suggest a key role for central neural processing; however, little is known about central neuroplasticity and DE. We used a model for tear deficient DE and assessed effects on eye blink behavior, orbicularis oculi muscle activity (OOemg), and trigeminal brainstem neural activity in male rats. Ocular-responsive neurons were recorded at the interpolaris/caudalis transition (Vi/Vc) and Vc/upper cervical cord (Vc/C1) regions under isoflurane, whereas OOemg activity was recorded under urethane. Spontaneous tear volume was reduced by ∼50% at 14 days after exorbital gland removal. Hypertonic saline–evoked eye blink behavior in awake rats was enhanced throughout the 14 days after surgery. Saline-evoked neural activity at the Vi/Vc transition and in superficial and deep laminae at the Vc/C1 region was greatly enhanced in DE rats. Neurons from DE rats classified as wide dynamic range displayed enlarged convergent periorbital receptive fields consistent with central sensitization. Saline-evoked OOemg activity was markedly enhanced in DE rats compared with controls. Synaptic blockade at the Vi/Vc transition or the Vc/C1 region greatly reduced hypertonic saline–evoked OOemg activity in DE and sham rats. These results indicated that persistent tear deficiency caused sensitization of ocular-responsive neurons at multiple regions of the caudal trigeminal brainstem and enhanced OOemg activity. Central sensitization of ocular-related brainstem circuits is a significant factor in DE and likely contributes to the apparent weak correlation between peripheral signs of tear dysfunction and symptoms of irritation.

Temporomandibular joint-evoked responses by spinomedullary neurons and masseter muscle are enhanced after repeated psychophysical stress

Psychological stress is a risk factor for the development of musculoskeletal pain of the head and neck; however, the basis for this relationship remains uncertain. This study tested the hypothesis that psychophysical stress alone was sufficient to alter the encoding properties of spinomedullary dorsal horn neurons and masseter muscle activity in male rats. Repeated forced swim conditioning increased markedly both the background firing rate and temporomandibular joint (TMJ)‐evoked activity of neurons in deep dorsal horn, while neurons in superficial laminae were less affected. Stress also increased the responses to stimulation of facial skin overlying the TMJ of neurons in deep and superficial dorsal horn. TMJ‐evoked masseter muscle activity was enhanced significantly in stressed rats, an effect that was reduced by prior blockade of the spinomedullary junction region. These data indicated that repeated psychophysical stress induced widespread effects on the properties of medullary dorsal horn neurons and masseter muscle activity. The effects of stress were seen preferentially on neurons in deep dorsal horn and included enhanced responses to chemosensory input from the TMJ and mechanical input from overlying facial skin. The stress‐induced elevation in TMJ‐evoked masseter muscle activity matched well with the changes seen in dorsal horn neurons. It is concluded that the spinomedullary junction region plays a critical role in the integration of psychophysical stress and sensory information relevant for nociception involving deep craniofacial tissues.

Estrogen status and psychophysical stress modify temporomandibular joint input to medullary dorsal horn neurons in a lamina-specific manner in female rats

&NA; Estradiol and behavioral stress modify temporomandibular joint‐evoked trigeminal caudalis neural activity in a lamina‐specific manner and enhance jaw muscle activity in female rats. &NA; Estrogen status and psychological stress contribute to the expression of several chronic pain conditions including temporomandibular muscle and joint disorders (TMJD). Sensory neurons that supply the temporomandibular joint (TMJ) region terminate in laminae I and V of the spinal trigeminal nucleus (Vc/C1–2 region); however, little is known about lamina‐specificity and environmental influences on the encoding properties of TMJ brainstem neurons. To test the hypothesis that Vc/C1–2 neurons integrate both interoceptive and exteroceptive signals relevant for TMJ nociception, we recorded TMJ‐evoked activity in superficial and deep laminae of ovariectomized rats under high and low estradiol (E2) and stress conditions. Rats received daily injections of low (LE) or high (HE) dose E2 and were subjected to forced swim (FS) or sham swim conditioning for 3 days. The results revealed marked lamina‐specificity in that HE rats displayed enhanced TMJ‐evoked activity in superficial, but not deep, laminae independent of stress conditioning. By contrast, FS conditioned rats displayed increased background firing and TMJ‐evoked activity of neurons in deep, but not superficial, laminae independent of E2 status. FS also enhanced TMJ‐evoked masseter muscle activity and suggested the importance of deep dorsal horn neurons in mediating evoked jaw muscle activity. In conclusion, E2 status and psychophysical stress play a significant role in modifying the encoding properties of TMJ‐responsive medullary dorsal horn neurons with a marked lamina‐specificity.

EVIDENCE FOR TRPA1 INVOLVEMENT IN CENTRAL NEURAL MECHANISMS IN A RAT MODEL OF DRY EYE

Dry eye (DE) disease is commonly associated with ocular surface inflammation, an unstable tear film and symptoms of irritation. However, little is known about the role of central neural mechanisms in DE. This study used a model for persistent aqueous tear deficiency, exorbital gland removal, to assess the effects of mustard oil (MO), a transient receptor potential ankyrin (TRPA1) agonist, on eyeblink and eyewipe behavior and Fos-like immunoreactivity (Fos-LI) in the trigeminal brainstem of male rats. Spontaneous tear secretion was reduced by about 50% and spontaneous eyeblinks were increased more than 100% in DE rats compared to sham rats. MO (0.02-0.2%) caused dose-related increases in eyeblink and forelimb eyewipe behavior in DE and sham rats. Exorbital gland removal alone was sufficient to increase Fos-LI at the ventrolateral pole of trigeminal interpolaris/caudalis (Vi/Vc) transition region, but not at more caudal regions of the trigeminal brainstem. Under barbiturate anesthesia ocular surface application of MO (2-20%) produced Fos-LI in the Vi/Vc transition, in the mid-portions of Vc and in the trigeminal caudalis/upper cervical spinal cord (Vc/C1) region that was significantly greater in DE rats than in sham controls. MO caused an increase in Fos-LI ipsilaterally in superficial laminae at the mid-Vc and Vc/C1 regions in a dose-dependent manner. Smaller, but significant, increases in Fos-LI also were seen in the contralateral Vc/C1 region in DE rats. TRPA1 protein levels in trigeminal ganglia from DE rats ipsilateral and contralateral to gland removal were similar. Persistent tear reduction enhanced the behavioral and trigeminal brainstem neural responses to ocular surface stimulation by MO. These results suggested that TRPA1 mechanisms play a significant role in the sensitization of ocular-responsive trigeminal brainstem neurons in this model for tear deficient DE.

Posterior hypothalamic modulation of light-evoked trigeminal neural activity and lacrimation

Enhanced light sensitivity is a common feature of many neuro-ophthalmic conditions and some chronic headaches. Previously we reported that the bright light-evoked increases in trigeminal brainstem neural activity and lacrimation depended on a neurovascular link within the eye (Okamoto et al., 2012). However, the supraspinal pathways necessary for these light-evoked responses are not well defined. To assess the contribution of the posterior hypothalamic area (PH), a brain region closely associated with control of autonomic outflow, we injected bicuculline methiodide (BMI), a GABAa receptor antagonist, into the PH and determined its effect on the encoding properties of ocular neurons at the ventrolateral trigeminal interpolaris/caudalis transition (Vi/Vc) and caudalis/upper cervical cord junction (Vc/C1) regions and on reflex lacrimation in male rats under isoflurane anesthesia. BMI markedly reduced light-evoked (>80%) responses of Vi/Vc and Vc/C1 neurons at 10 min with partial recovery by 50 min after injection. BMI also reduced (>35%) the convergent cutaneous receptive field area of Vi/Vc and Vc/C1 ocular neurons indicating that both intra-ocular and periorbital cutaneous inputs were affected by changes in PH outflow. Light-evoked lacrimation was reduced by >35% at 10 min after BMI, while resting mean arterial pressure increased promptly and remained elevated (>20 mmHg) throughout the 50-min post-injection period. These results suggested that PH stimulation, acting in part through increased sympathetic activity, significantly inhibited light- and facial skin-evoked activity of ocular neurons at the Vi/Vc and Vc/C1 region. These data provide further support for the hypothesis that autonomic outflow plays a critical role in mediating light-evoked trigeminal brainstem neural activity and reflex lacrimation.

Trigeminal pathways for hypertonic saline- and light-evoked corneal reflexes

Cornea-evoked eyeblinks maintain tear film integrity on the ocular surface in response to dryness and protect the eye from real or potential damage. Eyelid movement following electrical stimulation has been well studied in humans and animals; however, the central neural pathways that mediate protective eyeblinks following natural nociceptive signals are less certain. The aim of this study was to assess the role of the trigeminal subnucleus interpolaris/caudalis (Vi/Vc) transition and subnucleus caudalis/upper cervical cord (Vc/C1) junction regions on orbicularis oculi electromyographic (OOemg) activity evoked by ocular surface application of hypertonic saline or exposure to bright light in urethane anesthetized male rats. The Vi/Vc and Vc/C1 regions are the main sites of termination for trigeminal afferent nerves that supply the ocular surface, while hypertonic saline (saline=0.15-5M) and bright light (light=5k-20klux) selectively activate ocular surface and intraocular trigeminal nerves, respectively, and excite second-order neurons at the Vi/Vc and Vc/C1 regions. Integrated OOemg activity, ipsilateral to the applied stimulus, increased with greater stimulus intensities for both modalities. Lidocaine applied to the ocular surface inhibited OOemg responses to hypertonic saline, but did not alter the response to light. Lidocaine injected into the trigeminal ganglion blocked completely the OOemg responses to hypertonic saline and light indicating a trigeminal afferent origin. Synaptic blockade by cobalt chloride of the Vi/Vc or Vc/C1 region greatly reduced OOemg responses to hypertonic saline and bright light. These data indicate that OOemg activity evoked by natural stimuli known to cause irritation or discomfort in humans depends on a relay in both the Vi/Vc transition and Vc/C1 junction regions.

Increased Neopterin Levels in Brains of Patients with Human Immunodeficiency Virus Type 1 Infection

Abstract: Postmortem levels of native neopterin (D‐erythroneopterin) were measured in cerebral cortical samples from 44 human immunodeficiency virus type 1‐infected and eight uninfected, nonneurological control patients. Cerebral cortical gray and white matter neopterin levels for the controls ranged from 0.5 to 7.2 pmol/mg of protein in contrast to neopterin levels in brains of the virus‐infected patients, which frequently were more than threefold and occasionally more than 30‐fold higher than mean control levels. Cortical neopterin levels did not correlate with severity of the acquired immunodeficiency syndrome dementia complex, but subcortical levels correlated with the presence of active human immunodeficiency virus type 1 infection, as reflected by pathological evidence of multinucleated giant cell encephalitis. Evidence of opportunistic cytomegalovirus infections in ∼25% of the human immunodeficiency virus type 1‐infected patients was associated with enhanced levels of neopterin in frontal cortex.